Amphiphilic hyperbranched fluoropolymers as nanoscopic 19F magnetic resonance imaging agent assemblies

Three hyperbranched fluoropolymers were synthesized and their micelles were constructed as potential (19)F MRI agents. A hyperbranched star-like core was first synthesized via atom transfer radical self-condensing vinyl (co)polymerization (ATR-SCVCP) of 4-chloromethyl styrene (CMS), lauryl acrylate (LA), and 1,1,1-tris(4'-(2'-bromoisobutyryloxy)phenyl)ethane (TBBPE). The polymerization gave a small core with M n of 5.5 kDa with PDI of 1.6, which served as a macroinitiator. Trifluoroethyl methacrylate (TFEMA) and tert-butyl acrylate (tBA) in different ratios were then "grafted" from the core to give three polymers with M(n) of about 120 kDa and PDI values of about 1.6-1.8. After acidolysis of the tert-butyl ester groups, amphiphilic, hyperbranched star-like polymers with M(n) of about 100 kDa were obtained. These structures were subjected to micelle formation in aqueous solution to give micelles having TEM-measured diameters ranging from 3-8 nm and DLS-measured hydrodynamic diameters from 20-30 nm. These micelles gave a narrow, single resonance by (19)F NMR spectroscopy, with a half-width of approximately 130 Hz. The T1/T2 parameters were about 500 and 50 ms, respectively, and were not significantly affected by the composition and sizes of the micelles. (19)F MRI phantom images of these fluorinated micelles were acquired, which demonstrated that these fluorinated micelles maybe useful as novel (19)F MRI agents for a variety of biomedical studies.     

嵌段共聚物聚乙二醇-聚苹果酸苄基酯载药胶束的制备及性质研究

目的：为构建聚合物胶束药物运载体系,制备嵌段共聚物聚乙二醇-聚苹果酸苄基酯载药胶束并测定其性质。方法：以L-天冬氨酸为原料,重氮化、环化后经开环聚合得到聚苹果酸苄基酯。氨基聚乙二醇通过酰胺键连接到β-聚苹果酸苄基酯上形成两亲性嵌段共聚物,喜树碱做药物模型制备载药胶束。动态光散射法测定胶束粒径、评价胶束稳定性,高效液相法测定喜树碱载药率和包封率,芘荧光法与动态光散射法测定临界胶束浓度。结果：喜树碱包封率72%,载药率6%,临界胶束浓度为40μg.mL-1。随着聚苹果酸苄基酯分子量减小,胶束稳定性增强。结论：聚乙二醇-聚苹果酸苄基酯在疏水链/亲水链分子量比值为2-4时在水中可自组装形成纳米胶束,可作为性能优良的聚合物药物载体。     

Synthesis of well-defined amphiphilic block copolymers having phospholipid polymer sequences as a novel biocompatible polymer micelle reagent

To realize safer and effective drug administration, novel well-defined and biocompatible amphiphilic block copolymers containing phospholipid polymer sequences were synthesized. At first, the homopolymer of 2-methacryloyloxyethylphosphorylcholine (MPC) was synthesized in water by reversible addition-fragmentation chain transfer (RAFT) controlled radical polymerization. The "living" polymerization was confirmed by the fact that the number-average molecular weight increased linearly with monomer conversion while the molecular weight distribution remained narrow independent of the conversion. The poly(MPC) thus prepared is end-capped with a dithioester moiety. Using the dithioester-capped poly(MPC) as a macro chain transfer agent, AB diblock copolymers of MPC and n-butyl methacrylate (BMA) were synthesized. Associative properties of the amphiphilic block copolymer (pMPC(m)-BMA(n)) with varying poly(BMA) block lengths were investigated using NMR, fluorescence probe, static light scattering (SLS), and quasi-elastic light scattering (QELS) techniques. Proton NMR data in D2O indicated highly restricted motions of the n-butyl moieties, arising from hydrophobic associations of poly(BMA) blocks. Fluorescence spectra of N-phenyl-1-naphthylamine indicated that the probes were solubilized in the polymer micelles in water. The formation of polymer micelles comprising a core with poly(BMA) blocks and shell with hydrophilic poly(MPC) blocks was suggested by SLS and QELS data. The size and mass of the micelle increased with increasing poly(BMA) block length. With an expectation of a pharmaceutical application of pMPC(m)-BMA(n), solubilization of a poorly water-soluble anticancer agent, paclitaxel (PTX), was investigated. PTX dissolved well in aqueous solutions of pMPC(m)-BMA(n) as compared with pure water, implying that PTX is incorporated into the hydrophobic core of the polymer micelle. Since excellent biocompatible poly(MPC) sequences form an outer shell of the micelle, pMPC(m)-BMA(n) may find application as a promising reagent to make a good formulation with a hydrophobic drug.     

Glucose-responsive vehicles containing phenylborate ester for controlled insulin release at neutral pH

This study is devoted to developing amphiphilic block polymers based on phenylborate ester, which can self-assemble to form nanoparticles, as a glucose-sensitive drug carrier. Poly(ethylene glycol)-block-poly[(2-phenylboronic esters-1,3-dioxane-5-ethyl) methylacrylate] (MPEG5000-block-PBDEMA) was fabricated with MPEG5000-Br as a macroinitiator via atom transfer radical polymerization (ATRP). Using the solvent evaporation method, these block polymers can disperse in aqueous milieu to self-assemble into micellar aggregates with a spherical core-shell structure. Zeta potential and fluorescence techniques analysis showed a good purification effect, high encapsulation efficiency, and loading capacity of fluorescein isothiocyanate (FITC)-insulin-loaded polymeric micelles under optimal conditions. The in vitro insulin release profiles revealed definite glucose-responsive behavior of the polymeric micelles at pH 7.4 and 37 °C, depending on the environmental glucose concentration and the chemical composition of the block polymers. Further, circular dichroism spectroscopy demonstrated that the overall tertiary structure of the released insulin was in great agreement with standard insulin. (1)H NMR results of the polymeric micelles during glucose-responsive process supposed one possible insulin release mechanism via the polymer polarity transition from amphiphilic to double hydrophilic. The analysis of L929 mouse fibroblast cells viability suggested that the polymeric micelles from MPEG5000-block-PPBDEMA had low cell toxicity. The block polymers containing phenylborate ester that responded to changes in the glucose concentration at neutral pH are being aimed for use in self-regulated insulin delivery.     

Fabrication of multiresponsive shell cross-linked micelles possessing pH-controllable core swellability and thermo-tunable corona permeability

A double hydrophilic ABC triblock copolymer, poly(2-(diethylamino)ethyl methacrylate)-b-poly(2-(dimethylamino)ethyl methacrylate)-b-poly(N-isopropylacrylamide) (PDEA-b-PDMA-b-PNIPAM), containing the well-known pH-responsive PDEA block and thermoresponsive PNIPAM block, was synthesized by atom transfer radical polymerization via sequential monomer addition using ethyl 2-chloropropionate as the initiator. The obtained triblock copolymer exhibits interesting "schizophrenic" micellization behavior in aqueous solution, and supramolecularly self-assembles into three-layer "onion-like" PNIPAM-core micelles at acidic pH's and elevated temperatures and PDEA-core micelles with "inverted" structures at alkaline pH's and room temperature. In both cases, dynamic laser light scattering (LLS) and optical transmittance reveal the presence of near-monodisperse micelles, and the micelle formation/inversion process is fully reversible. Novel shell cross-linked (SCL) micelles with pH-responsive PDEA cores and thermoresponsive PNIPAM coronas were then facilely fabricated from the PDEA-b-PDMA-b-PNIPAM triblock copolymer by cross-linking the PDMA inner shells with 1,2-bis(2-iodoethoxy)ethane. The reversible pH-dependent swelling/shrinking of PDEA cores and thermosensitive collapse/aggregation of PNIPAM coronas of the obtained SCL micelles were investigated in detail by dynamic LLS, optical transmittance, and transmission electron microscopy. As the structurally stable SCL micelles possess pH-controllable core swellability and thermo-tunable corona permeability, the release profile of a model hydrophobic drug, dipyridamole, initially loaded within the hydrophobic PDEA core, can be dually controlled by both the solution pH and the temperature. This represents the first report of SCL micelles with multiresponsive cores and coronas, which may find practical applications in fields such as drug delivery and smart release.     

Polymer-based stimuli-responsive nanosystems for biomedical applications

The application of organic polymers and inorganic/organic hybrid systems in numerous fields of biotechnology has seen a considerable growth in recent years. Typically, organic polymers with diverse structures, compositional variations and differing molecular weights have been utilized to assemble polymeric nanosystems such as polymeric micelles, polymersomes, and nanohydrogels with unique features and structural properties. The architecture of these polymeric nanosystems involves the use of both hydrophobic and hydrophilic polymeric blocks, making them suitable as vehicles for diagnostic and therapeutic applications. Recently, “smart” or “intelligent” polymers have attracted significant attention in the biomedical field wherein careful introduction of specific polymeric modalities changes a banal polymeric nanosystem to an advanced stimuli-responsive nanosystem capable of performing extraordinary functions in response to an internal or external trigger such as pH, temperature, redox, enzymes, light, magnetic, or ultrasound. Further, incorporation of inorganic nanoparticles such as gold, silica, or iron oxide with surface-bound stimuli-responsive polymers offers additional advantages and multifunctionality in the field of nanomedicine. This review covers the physical properties and applications of both organic and organic/inorganic hybrid nanosystems with specific recent breakthroughs in drug delivery, imaging, tissue engineering, and separations and provides a brief discussion on the future direction.     

Potential of amphiphilically modified low molecular weight chitosan as a novel carrier for hydrophobic anticancer drug: Synthesis,characterization, micellization and cytotoxicity evaluation

Amphiphilically modified low molecular weight chitosan (LMWC) with long chain alkyl groups as hydrophobic moieties and carboxymethyl groups as hydrophilic moieties (N-octyl-N,O-carboxymethyl LMWC, OC-LMWC) was synthesized. Self-assembled polymeric micelles of OC-LMWC were prepared in aqueous environment. Critical micelle concentrations (CMC) of OC-LMWCs were varied from 8.7 to 27.7 mg/l. Paclitaxel (PTX) was successfully encapsulated into the hydrophobic cores of the nanoparticles. The drug loading content and entrapment efficiency were higher to 32.17% (w/w) and 80.61%, respectively. Differential scanning calorimetry (DSC), transmission electron microscope (TEM) observation and dynamic light scattering (DLS) measurements were carried out to determination the physicochemical properties of the micelles. MTT assay showed that the in vitro cytotoxic effect of the PTX-loaded micelles was comparable to that of the commercial formulation, but the blank micelles were far less than the Cremophor EL^® vehicle. These results suggested that OC-LMWC micelles were promising carriers for hydrophobic anticancer agents.     

Polymer terminal group effects on properties of thermoresponsive polymeric micelles with controlled outer-shell chain lengths

Well-defined amphiphilic diblock copolymers comprising thermoresponsive polymer segments of poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (PID) and hydrophobic polymer segments, poly(benzyl methacrylate) (PBzMA), were synthesized by controlled living radical polymerization. Terminal derivatization of PID segments to either hydroxyl or phenyl groups was achieved through reactions of coupling agents with thiol groups exposed by cleavage of terminal dithiobenzoate groups. Diblock copolymers formed core-shell type polymeric micelles with thermoresponsive outer shells. Hydrodynamic micellar diameters ranged from 12 to 31 nm, controlled by varying PID chain lengths. Differences in PID terminal groups did not affect the critical micelle concentration or micellar diameters. However, these groups demonstrated a significant influence on the micellar thermoresponses. Hydroxylated PID/PBzMA micelles exhibited a phase transition of approximately 40 degrees C, independent of PID molecular weights. Even though molecular weights and compositions of PID chains were equivalent except for terminal groups, micelles having the outermost surface phenyl groups exhibited drastically lower phase transition temperature shifts, especially for micelles with low molecular weight PID chains.     

Solubilization and controlled release of a hydrophobic drug using novel micelle-forming ABC triblock copolymers

Amphiphilic ABC triblock copolymers composed of monomethoxy-capped poly(ethylene glycol) (MPEG), poly(2-(dimethylamino)ethyl methacrylate) (DMA), and poly(2-(diethylamino)ethyl methacrylate) (DEA) have been synthesized by atom transfer radical polymerization (ATRP). These copolymers dissolve molecularly in acidic aqueous media at room temperature due to protonation of the tertiary amine groups on the DMA and DEA residues. On adjusting the pH with base, micellization occurred at pH 8, with the water-insoluble, deprotonated DEA block forming the hydrophobic cores and the MPEG and DMA blocks forming the hydrophilic micellar coronas and inner shells, respectively. This pH-induced micellization has been exploited to develop a solvent-free protocol for drug loading. A model hydrophobic drug, dipyridamole (DIP), which dissolves in acid but is insoluble above pH 5.8, was incorporated into the micelles by increasing the pH of an aqueous drug/copolymer mixture to 9. Both the empty and the drug-loaded micelles were characterized by dynamic light scattering and fluorescence studies. The interaction of both pyrene and DIP with the MPEG-DMA-DEA micelles was studied by fluorescence; both compounds had relatively high partition coefficients into the micelles, 4.5 x 10(5) and 1.5 x 10(4), respectively. Intensity-average micelle diameters ranged from 20 to 90 nm, depending on the polymer composition and concentration. Shorter MPEG blocks (Mn = 2000) produced larger micelles than longer MPEG blocks (Mn = 5000) due to the shift in the hydrophilic-hydrophobic balance of the copolymer. Transmission electron microscopy studies of the drug-loaded micelles indicated spherical morphologies and reasonably uniform particle size distributions, which is in marked contrast to the needlelike morphology observed for pure DIP in the absence of the copolymer. Experiments on controlled release demonstrated that DIP-loaded MPEG-DMA-DEA micelles act as a drug carrier, giving slow release to the surrounding solution over a period of days. Rapid release can be triggered by reducing the pH to reverse the micellization.     

Amphiphilic core-shell nanocarriers based on hyperbranched poly(ester amide)-star-PCL: synthesis, characterization, and potential as efficient phase transfer agent

Amphiphilic biodegradable star-shaped polymer was conveniently prepared by the Sn(Oct)2-catalyzed ring opening polymerization of epsilon-caprolactone (CL) with hyperbranched poly(ester amide) (PEA) as a macroinitiator. Various monomer/initiator ratios were employed to vary the length of the PCL arms. (1)H NMR and FTIR characterizations showed the successful synthesis of star polymer with high initiation efficiency. SEC analysis using triple detectors, RI, light scattering, and viscosity confirmed the controlled manner of polymerization and the star architecture. Because of the hydrophilic PEA core and hydrophobic PCL shell, the obtained star polymers displayed inverted unimolecular micellar structure confirmed by dynamic light scattering. Three water soluble dyes, congo red, methyl orange, and bromophenol blue, were used to investigate the host-guest behavior of the micelles. It proved that the core-shell unimolecular reverse micelles were able to transport polar dyes from water to the organic phase with a high efficiency of up to 22.6 dyes per polymer, indicating a great potential of the micelles as drug carriers. The influence of arm length and core size on the load efficiency of the nanocarrier was also evaluated.     

Synthesis and micellization of amphiphilic brush-coil block copolymer based on poly(epsilon-caprolactone) and PEGylated polyphosphoester

A novel biodegradable amphiphilic brush-coil block copolymer consisting of poly(epsilon-caprolactone) and PEGylated polyphosphoester was synthesized by ring opening polymerization. The composition and structure of the copolymer were characterized by 1H NMR, 13C NMR, and FT-IR, and the molecular weight and molecular weight distribution were analyzed by gel permeation chromatograph (GPC) measurements to confirm the diblock structure. These amphiphilic copolymers formed micellar structures in water, and the critical micelle concentrations (CMCs) were around 10(-3) mg/mL, which was determined using pyrene as a fluorescence probe. Transmission electron microscopy (TEM) images showed that the micelles took an approximately spherical shape with core-shell structure, which was further demonstrated by laser light scattering (LLS) technique. The degradation behavior of the polymeric micelle was also investigated in the presence of Pseudomonas lipase and characterized by GPC measurement. Such polymer micelles from brush-coil block copolymers are expected to have wide utility in the field of drug delivery.     

The pH dependence of polymerization and bundling by the essential bacterial cytoskeletal protein FtsZ

There is a growing body of evidence that bacterial cell division is an intricate coordinated process of comparable complexity to that seen in eukaryotic cells. The dynamic assembly of Escherichia coli FtsZ in the presence of GTP is fundamental to its activity. FtsZ polymerization is a very attractive target for novel antibiotics given its fundamental and universal function. In this study our aim was to understand further the GTP-dependent FtsZ polymerization mechanism and our main focus is on the pH dependence of its behaviour. A key feature of this work is the use of linear dichroism (LD) to follow the polymerization of FtsZ monomers into polymeric structures. LD is the differential absorption of light polarized parallel and perpendicular to an orientation direction (in this case that provided by shear flow). It thus readily distinguishes between FtsZ polymers and monomers. It also distinguishes FtsZ polymers and less well-defined aggregates, which light scattering methodologies do not. The polymerization of FtsZ over a range of pHs was studied by right-angled light scattering to probe mass of FtsZ structures, LD to probe real-time formation of linear polymeric fibres, a specially developed phosphate release assay to relate guanosine triphosphate (GTP) hydrolysis to polymer formation, and electron microscopy (EM) imaging of reaction products as a function of time and pH. We have found that lowering the pH from neutral to 6.5 does not change the nature of the FtsZ polymers in solution--it simply facilitates the polymerization so the fibres present are longer and more abundant. Conversely, lowering the pH to 6.0 has much the same effect as introducing divalent cations or the FtsZ-associated protein YgfE (a putative ZapA orthologue in E. coli)--it stabilizes associations of protofilaments.     

Nanoscopic cationic methacrylate star homopolymers: synthesis by group transfer polymerization, characterization and evaluation as transfection reagents

Seven star polymers with degrees of polymerization (DPs) of the arms from 10 to 100 and dimensions in the nanometer range were prepared using sequential group transfer polymerization of 2-(dimethylamino)ethyl methacrylate (DMAEMA, hydrophilic positively ionizable monomer) and ethylene glycol dimethacrylate (hydrophobic neutral cross-linker). The polymers were characterized in tetrahydrofuran by gel permeation chromatography and static light scattering to determine the molecular weights and the weight-average number of arms for each sample. The number of arms of the star polymers varied from 20 to 72. Aqueous solutions of the star polymers were studied by turbidimetry, hydrogen ion titration, and dynamic light scattering to determine their cloud points, pKs, and hydrodynamic diameters. The cloud points of the larger star polymers, with arm DP 30-100, were found to be 29-34 degrees C, almost independent of the DP of the arms. Similarly, the pKs of all star polymers were calculated to range between 6.7 and 7.0, again independent of the arm DP. In contrast, the hydrodynamic diameters of the star polymers strongly depended on the DP of the arms. In particular, by increasing the DP of the arms from 20 to 100, the hydrodynamic diameters in water increased from 7 to 31 nm. All star polymers were evaluated for their ability to transfect human cervical HeLa cancer cells with the modified plasmid pRLSV40 with the enhanced green fluorescent protein as the reporter gene. Our results showed that as the DP of the arms of the DMAEMA star homopolymers increased from 10 to 100, the overall transfection efficiency decreased, with the star polymer with DP of the arms of 10 emerging as the best transfection reagent. Systematic variation of the amounts of star polymer and plasmid DNA used in the transfections led to an optimization of the performance of this star polymer, yielding overall transfection efficiencies of 15%, comparable to the optimum overall transfection efficiency of the commercially available transfection reagent SuperFect of 13%.     

Cationic amphiphilic model networks based on symmetrical ABCBA pentablock terpolymers: synthesis, characterization, and modeling

Eight isomeric networks based on equimolar terpolymers were synthesized using group transfer polymerization (GTP) and were characterized in terms of their swelling properties. Two hydrophilic monomers, the nonionic methoxy hexa(ethylene glycol) methacrylate (HEGMA) and the ionizable 2-(dimethylamino)ethyl methacrylate (DMAEMA), and a hydrophobic (nonionic) monomer, methyl methacrylate (MMA), were employed for the syntheses. 1,4-Bis(methoxytrimethylsiloxymethylene)cyclohexane (MTSMC) was used as the bifunctional GTP initiator, while ethylene glycol dimethacrylate (EGDMA) served as the cross-linker. Seven of the networks were model networks, six of which were based on the symmetrical pentablock terpolymers ABCBA, ACBCA, BACAB, BCACB, CBABC, and CABAC, whereas the seventh model network was based on the statistical terpolymer. The eighth network was a randomly cross-linked network based on the statistical terpolymer, prepared by the simultaneous quaterpolymerization of the three monomers and the cross-linker. The molecular weights and molecular weight distributions of the linear pentablock terpolymer precursors, as well as those of their homopolymer and ABA triblock copolymer precursors, were characterized by gel permeation chromatography (GPC) in tetrahydrofuran. The sol fraction of each network was measured and found to be relatively low. The aqueous degrees of swelling of all networks were found to increase at acidic pH due to the ionization of the DMAEMA tertiary amine units. The acidic degrees of swelling of the pentablock terpolymer networks were lower than those of their statistical counterparts due to microphase separation in the former type of networks, also confirmed by thermodynamic calculations and small-angle neutron scattering experiments.     

Solubilization of docetaxel in poly(ethylene oxide)-block-poly(butylene/styrene oxide) micelles

Poly(ethylene oxide)-block-poly(styrene oxide) (PEO-b-PSO) and PEO-b-poly(butylene oxide) (PEO-b-PBO) of different chain lengths were synthesized and characterized for their self-assembling properties in water by dynamic/static light scattering, spectrofluorimetry, and transmission electron microscopy. The resulting polymeric micelles were evaluated for their ability to solubilize and protect the anticancer drug docetaxel (DCTX) from degradation. The drug release kinetics as well as the cytotoxicity of the loaded micelles were assessed in vitro. All polymers formed micelles with a highly viscous core at low critical association concentrations (<10 mg/L). Micelle morphology depended on the nature of the hydrophobic block, with PBO- and PSO-based micelles yielding monodisperse spherical and cylindrical nanosized aggregates, respectively. The maximum solubilization capacity for DCTX ranged from 0.7 to 4.2% and was the highest for PSO micelles exhibiting the longest hydrophobic segment. Despite their high affinity for DCTX, PEO-b-PSO micelles were not able to efficiently protect DCTX against hydrolysis under accelerated stability testing conditions. Only PEO-b-PBO bearing 24 BO units afforded significant protection against degradation. In vitro, DCTX was released slower from the latter micelles, but all formulations possessed a similar cytotoxic effect against PC-3 prostate cancer cells. These data suggest that PEO-b-P(SO/BO) micelles could be used as alternatives to conventional surfactants for the solubilization of taxanes.     

The pH Dependence of Polymerization and Bundling by the Essential Bacterial Cytoskeltal Protein FtsZ

There is a growing body of evidence that bacterial cell division is an intricate coordinated process of comparable complexity to that seen in eukaryotic cells. The dynamic assembly of Escherichia coli FtsZ in the presence of GTP is fundamental to its activity. FtsZ polymerization is a very attractive target for novel antibiotics given its fundamental and universal function. In this study our aim was to understand further the GTP-dependent FtsZ polymerization mechanism and our main focus is on the pH dependence of its behaviour. A key feature of this work is the use of linear dichroism (LD) to follow the polymerization of FtsZ monomers into polymeric structures. LD is the differential absorption of light polarized parallel and perpendicular to an orientation direction (in this case that provided by shear flow). It thus readily distinguishes between FtsZ polymers and monomers. It also distinguishes FtsZ polymers and less well-defined aggregates, which light scattering methodologies do not. The polymerization of FtsZ over a range of pHs was studied by right-angled light scattering to probe mass of FtsZ structures, LD to probe real-time formation of linear polymeric fibres, a specially developed phosphate release assay to relate guanosine triphosphate (GTP) hydrolysis to polymer formation, and electron microscopy (EM) imaging of reaction products as a function of time and pH. We have found that lowering the pH from neutral to 6.5 does not change the nature of the FtsZ polymers in solution—it simply facilitates the polymerization so the fibres present are longer and more abundant. Conversely, lowering the pH to 6.0 has much the same effect as introducing divalent cations or the FtsZ-associated protein YgfE (a putative ZapA orthologue in E. coli)—it stablizes associations of protofilaments.     

Association behavior of biotinylated and non-biotinylated poly(ethylene oxide)-b-poly(2-(diethylamino)ethyl methacrylate)

Biotinylated and non-biotinylated copolymers of poly(ethylene oxide) (PEO) and poly(2-(diethylamino)ethyl methacrylate) (PDEAEMA) were synthesized by the atom transfer radical polymerization technique. The chemical compositions of the copolymers as determined by NMR are represented by PEO(113)PDEAEMA(70) and biotin-PEO(104)PDEAEMA(93), respectively. The aggregation behavior of these polymers in aqueous solutions at different pHs and ionic strengths was studied using a combination of potentiometric titration, dynamic light scattering, static light scattering, and transmission electron microscopy. Both PEO-b-PDEAEMA and biotin-PEO-b-PDEAEMA diblock copolymers form micelles at high pH with hydrodynamic radii (R(h)) of about 19 and 23 nm, respectively. At low pH, the copolymers are dispersed as unimers in solution with R(h) values of about 6-7 nm. However, at a physiological salt concentration (c(s)) of about 0.16 M NaCl and a pH of 7-8, the copolymers form large loosely packed Gaussian chains, which were not present at the low c(s) of 0.001 M NaCl. The critical micelle concentrations (cmc's) and the cytotoxicities of the copolymers were investigated to determine a suitable polymer concentration range for future biological applications. Both PEO-b-PDEAEMA and biotin-PEO-b-PDEAEMA diblock copolymers possess identical cmc values of about 0.0023 mg/g, while the cytotoxicity test indicated that the copolymers are not toxic up to 0.05 mg/g (>83% cell survival at this concentration).     

Oxime linkage: a robust tool for the design of pH-sensitive polymeric drug carriers

Oxime bonds dispersed in the backbones of the synthetic polymers, while young in the current spectrum of the biomedical application, are rapidly extending into their own niche. In the present work, oxime linkages were confirmed to be a robust tool for the design of pH-sensitive polymeric drug delivery systems. The triblock copolymer (PEG-OPCL-PEG) consisting of hydrophilic poly(ethylene glycol) (PEG) and hydrophobic oxime-tethered polycaprolactone (OPCL) was successfully prepared by aminooxy terminals of OPCL ligating with aldehyde-terminated PEG (PEG-CHO). Owing to its amphiphilic architecture, PEG-OPCL-PEG self-assembled into the micelles in aqueous media, validated by the measurement of critical micelle concentration (CMC). The MTT assay showed that PEG-OPCL-PEG exhibited low cytotoxicity against NIH/3T3 normal cells. Doxorubicin (DOX) as a model drug was encapsulated into the PEG-OPCL-PEG micelles. Drug release study revealed that the DOX release from micelles was significantly accelerated at mildly acid pH of 5.0 compared to physiological pH of 7.4, suggesting the pH-responsive feature of the drug delivery systems with oxime linkages. Flow cytometry and confocal laser scanning microscopy (CLSM) measurements indicated that these DOX-loaded micelles were easily internalized by living cells. MTT assay against HeLa cancer cells showed DOX-loaded PEG-OPCL-PEG micelles had a high anticancer efficacy. All of these results demonstrate that these polymeric micelles self-assembled from oxime-tethered block copolymers are promising carriers for the pH-triggered intracellular delivery of hydrophobic anticancer drugs.     

Formation of core-shell type biodegradable polymeric micelles from amphiphilic poly(aspartic acid)-block-polylactide diblock copolymer

Poly(aspartic acid)-block-polylactide diblock copolymers (PAsp-b-PLAs) having both hydrophilic and hydrophobic segments of various lengths were synthesized. These PAsp-b-PLA diblock copolymers formed polymeric micelles consisting of a hydrophobic PLA core and a hydrophilic, pH-sensitive PAsp shell in aqueous solution. The effects of the segment length of both the PLA and the PAsp portions and the pH of the solution on the shapes and sizes of the PAsp-b-PLA polymeric micelles were investigated. The results indicated a balance between the effects of electrostatic repulsion, hydrogen bonding in the PAsp shell layer, and hydrophobic interactions in the PLA core determine the sizes of the PAsp-b-PLA polymeric micelles. Moreover, the PAsp-b-PLA polymeric micelles did not possess any cytotoxic activity against L929 fibroblast cells. The obtained polymeric micelle should be useful for biodegradable biomedical materials such as drug delivery vehicle.     

Characteristics of the aggregative state of the substrate in the reaction of 5-lipoxygenase oxidation of linoleic acid

5-lipoxygenase (EC 1.13.11.12) oxidizes polyunsaturated fatty acids by molecular oxygen. The enzyme acts in close contact with the cell membranes, which main components are ionic and non-ionic lipids. In order to investigate the kinetic parameters of 5-lipoxygenase reaction in vitro, extremely hydrophobic fatty acid substrate (linoleic acid) should be solubilized in the reaction mixture. We used Lubrol PX ("Sigma" Chem. Co), as a non-ionic detergent consisted of oligoethylene glycol and fatty alcohol. Linoleic acid and Lubrol PX formed mixed micelles thus solubilizing the fatty acid substrate in a buffer with appropriate pH. We have studied the sizes and shapes of mixed micelles Lubrol PX/linoleic acid (aggregates type 1) and Lubrol PX/linoleic acid/SDS (aggregates type 2; SDS was an effective activator of potato tuber 5-lipoxygenase) by means of gel-filtration and laser light scattering techniques. The parameters under investigation were molecular weights, Stocks radii and shapes of the mixed micelles. The average molecular weights and Stocks radii of the mixed micelles type 1 determined by mean of gel-filtration on Sephadex G-200 were 95,142 +/- 5184 Da and 3.45 +/- 0.11 nm, respectively. The same parameters for the mixed micelles type 2 were 73,694 +/- 893 Da and 3.02 +/- 0.02 nm, respectively. The strong similarity in physicochemical parameters for both types of mixed micelles indicated that SDS did not influence the size and shape of mixed micelles of Lubrol PX and linoleic acid. The activatory action of SDS on potato tuber lipoxygenase may be a result of electrostatic effect or direct participation of SDS in enzymatic catalysis. The laser light scattering technique allowed to determine two main fraction of particles in type 1 system with hydrodynamic diameters 2.6 and 5.7 nm and relative contribution to light scattering 13 and 87%, respectively. The particles with d = 5.7 nm were interpreted as the mixed micelles. The particles with d = 2.6 nm were interpreted as isolated molecules of Lubrol PX, linoleic acid and (or) their premicellar aggregates. The data obtained are to be used in creation of reliable physical and mathematical models of 5-lipoxygenase.